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Mtor-Specific Inhibitors in BRAF-Mutant Melanoma Cells Counteracted by Dual Pathway Blockade

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Mtor-Specific Inhibitors in BRAF-Mutant Melanoma Cells Counteracted by Dual Pathway Blockade www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 4 Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/ mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade Ilaria Penna1,2, Alessandra Molla1,2, Giulia Grazia1,2, Loredana Cleris2, Gabriella Nicolini1,2, Federica Perrone3, Benedetta Picciani3, Michele Del Vecchio4, Filippo de Braud4, Roberta Mortarini1,2,*, Andrea Anichini1,2,* 1Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3Laboratory of Molecular Pathology, Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4Medical Oncology Unit 1, Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy *These authors contributed equally to this work Correspondence to: Andrea Anichini, e-mail: [email protected] Keywords: melanoma, BRAF, MEK, PI3K/mTOR, apoptosis Received: June 30, 2015 Accepted: November 22, 2015 Published: December 14, 2015 ABSTRACT Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/ mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross- resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/ mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype. www.impactjournals.com/oncotarget 3947 Oncotarget INTRODUCTION inhibitors. Extensive drug interaction analysis on all 49 cell lines and mechanistic studies in cross-resistant cell lines Inhibitors of BRAFV600E have changed the clinical indicated that co-targeting of MEK1/2 and PI3K/mTOR, management of patients with BRAF-mutant advanced in-vitro and in-vivo, was a more effective combinatorial melanoma, since significant improvements in progression- treatment, compared to co-targeting of BRAF and PI3K/ free survival (PFS) and in overall survival (OS) have been mTOR, to counteract the primary cross-resistant phenotype. reported in Phase 3 trials [1–2]. These inhibitors can induce objective responses or stabilization of disease in RESULTS a high fraction of patients [2–3], although relapse occurs due to adaptive [4] or acquired [5] resistance mechanisms. Primary resistance to BRAFV600E inhibition In addition, ~20% of patients show primary/intrinsic is associated with cross-resistance to MEK1/2 resistance to BRAF inhibitors and experience tumor and PI3K/mTOR inhibitors in BRAF-mutant progression at first assessment during therapy [2–3]. melanoma cells Lack of response has been observed even in trials with MEK inhibitors as Trametinib and Selumetinib/AZD6244 We used 49 BRAF-mutant melanoma cell lines [6–7], pointing to primary resistance also to the targeting isolated from surgical specimens of patients not previously of MEK. To counteract resistance to BRAF inhibitors, treated with BRAFV600E inhibitors, nor with any several clinical trials based on combinatorial targeting other target-specific inhibitor, to test responsiveness to of BRAF and MEK have been carried out recently, and BRAFV600E (PLX4720), MEK1/2 (AZD6244), dual results have indicated a significant improvement in PFS PI3K/mTOR (BEZ235) and dual mTORC1/2 (AZD8055) and (in one study) in OS, compared to monotherapy with inhibitors (Figure 1A). Three susceptibility groups were the BRAF inhibitor alone [8–10]. However, strikingly, in defined by ranking cell lines based on their IC50 values for all these studies ~30% of the patients showed progressive PLX4720. Strong resistance (IC50 > 1 μM) was found in disease at 6 months of treatment [8–10], pointing to the 21/49 cell lines (42.9%, group 1), intermediate resistance potential role of cross-resistance mechanisms. Indeed, (IC50 = 0.1 to 1 μM) and susceptibility (IC50 < 0.1 μM) to primary cross-resistance to BRAF and MEK inhibitors has PLX4720 were observed in 16/49 (32.7%, group 2) and been documented in a subset of melanomas, where it is 12/49 cell lines (24.5%, group 3), respectively (Figure 1A related to the MITF profile [11–13], and in cell lines [14]. and Supplementary Table 1D for descriptive statistics). In the attempt to address all these limitations Seventeen out of 21 melanoma lines (marked with an associated with targeting of a single (MAPK) pathway, “x” in Figure 1A) classified in group 1, showed either several pre-clinical studies have suggested that combi- strong or intermediate primary resistance to MEK1/2 and natorial targeting of MAPK and PI3K/mTOR intracellular PI3K/mTOR inhibitors. Strong or intermediate cross- pathways may be a potential approach to improve target resistance to PI3K/mTOR inhibitors was also found in therapy of melanoma and to overcome resistance and 11/16 cell lines in group 2, while only two cell lines in cross-resistance mechanisms [14–17]. However, key group 3 (PLX4720 susceptible) showed strong resistance questions to be answered are whether BRAF-mutated to the PI3K/mTOR inhibitors (Figure 1A). Hierarchical melanomas, with intrinsic resistance to BRAF and/or clustering of log-transformed and normalized IC50 values MEK inhibitors, also have primary cross-resistance to confirmed the existence of distinct subsets of cell lines PI3K/mTOR inhibitors and which is the frequency of such with cross-resistance to all inhibitors or susceptible to cross-resistant phenotype. A second issue to be addressed all of them (Figure 2A). Spearman correlation analysis is whether combinatorial targeting of different signaling of IC50 values for all six possible combinations of four pathways is effective even in the tumors with primary inhibitors showed that all the susceptibility profiles were cross-resistance. significantly correlated (Figure 2B). Recently, we observed instances of primary cross- To validate these findings, an independent panel of resistance to a MEK1/2 (AZD6244) and to a dual PI3K/ short-term melanoma cell cultures (all tested between the mTOR (BEZ235) inhibitor in some melanoma cell lines third and fifth in-vitro passage), obtained from 23 BRAF- (18). Starting from this initial evidence, in this study we mutant metastatic specimens of patients not previously assessed responsiveness to BRAFV600E-, MEK1/2-, treated with target-specific inhibitors, was used to test dual PI3K/mTOR- and dual mTORC1/2-specific inhibitors responsiveness to the same set of inhibitors. The same in 49 BRAF-mutant melanoma cell lines and in an classification into three subsets based on ranking of independent set of 23 BRAF-mutant short-term melanoma PLX4720 IC50 values was applied. We found that 6/6 cell cultures, all isolated from patients never treated with PLX4720-resistant melanoma cell cultures (group 1) target therapy. Among cell lines with strong intrinsic showed strong (i.e. IC50 > 1 μM) or intermediate resistance to the BRAF inhibitor (IC50 > 1 μM, n = 21), (i.e. IC50 > 0.1 μM) cross-resistance to MEK1/2 and 81% (seventeen) showed strong or intermediate cross- PI3K/mTOR inhibitors, and 11/13 cultures in group 2 resistance to the MEK1/2- and the PI3K/mTOR-specific (intermediate resistance to PLX4720) showed also strong www.impactjournals.com/oncotarget 3948 Oncotarget Figure 1: Responsiveness to BRAF-V600E-, MEK1/2- or PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cell lines. (A) Susceptibility to PLX4720 (BRAFV600E inhibitor), AZD6244 (MEK1/2 inhibitor), BEZ235 (dual PI3K/mTOR inhibitor) and AZD8055 (dual mTORC1/2 inhibitor), shown as IC50 values (μM), was assessed by a 72 h MTT assay in a panel of 49 melanoma cell lines. IC50 values obtained through non linear regression analysis of twelve-point dose-response curves spanning 4 logs of inhibitor concentrations. Melanomas were distinguished into three groups after ranking based on PLX4720 IC50 values (group 1: IC50 > 1 μM; group 2: IC50 > 0.1 μM but < 1 μM; group 3: IC50 ≤ 0.1 μM). (B) Color code used for highlighting differences in IC50 values in panel A. www.impactjournals.com/oncotarget 3949 Oncotarget
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